Materials and Methods

Preparation of PCR master mix

Sample from practical 2 (pUC19 vector plasmid) was taken out from the freezer and was keep on ice thought
out the experiment. Other PCR components such as primers, Taq polymerase, dNTPs, 5x buffer, and MgCl 2
was also keep on ice thought out the experiment. The calculation for PCR master mix was calculated
beforehand (as shown in results). The sterile distilled water used was prepared in individual ependoft tube to
prevent contamination. The PCR master mix was calculated for 4 reaction tube. Each of the reaction tube
was label according to the content, DNA sample, - , + , and control. After the master mix was prepared, then
aliquot to individual reaction tube according to the label. One has remember to include DNA sample, Taq
polymerase and primer lastly or the moment before running the PCR. The amount of sterile distilled water to
be added to the reaction is based on the calculation of all the amount of the components of the master mix
minus total volume.
Preparation of agarose gel
The agarose gel was prepared with 0.8 % concentration. The dilution of 10X TBE to 1X TBE was done by
diluting 100 ml of 10X TBE buffer to 400 ml sterile distilled water. 0.8g of agarose powder was weighted
and diluted in 100ml of 1X TBE buffer then heated in microwave oven for 1 minute or until the powder is
completely dissolved and has clear appearance. A volume of 100l of 1000X GelRed stain was added into
the gel slurry once the gel was cold to touch (approximately 45C). The gel slurry was casted in the gel cast
of electrophoresis set with comb in position. The gel slurry was poured up to half of the comb heigh and let it
harden. If there is a presence of air bubble during the casting of the gel, the bubble need to be removed using
the pippete tip.
Running the PCR
The PCR was run according to the following conditions :
Step

Temperature (C)

Time (min)

Initial denaturation

95

Denaturation

95

0.5

Annealing

55

0.5

Extension

72

Final extension

72

10

The PCR reaction was set to repeat for 35 times. After the completion of the PCR reaction, the sample was
stored in -20C in the freezer for next week.